DESCRIPTION: Ischemic heart disease is the primary cause of death in both men and women globally. Ischemic heart conditions, including angina and myocardial infarction, are characterized by loss of blood flow to the heart tissue and reduced cardiac function, both reducing quality of life and increasing mortality. Improved recovery of cardiac function following a myocardial infarction has been observed in several studies upon the injection of high concentrations of endothelial progenitor cells (EPCs) to the ischemic area. Multiple types of EPCs have been shown to play pivotal roles in the formation of new blood vessels, and are promising candidates for accelerated and increased repair of ischemic tissues. Follow up studies indicate both high purity and quantity of EPCs are critical to recovery of function, yet current technologies for sorting of EPCs are incapable of isolating these cells in the purity and speed required for widespread clinical use. This research grant will develop a novel technique for the isolation of EPCs which yields a high-purity isolate at high throughput. The developed technology, Antigen Specific Lysis (ASL), is capable of being performed in a typical research lab setting where batches exceeding 200 mL (>1010 cells) are completed in under an hour. The proposed work will develop high speed selection modalities for the isolation of high purity, functional EPCs. ASL uses targeted coatings to selectively protect EPCs while all other cells are lysed. Preliminary results demonstrate 1) antibody- initiator conjugates are capable of selectively forming a ~100 nm polymer coating on the exterior of only antigen expressing cells, 2) these coatings stabilize cells in harsh environments (5% SDS), and 3) viable cells are released at high purity (>99% purity for antigen positive cells spiked into blood). This research grant drives new advances in wavelength specific polymerization and degradation for the selective protection of targeted cells and the subsequent release of cells with increased viability and proliferative capacity. Sequential selection for CD133+ and CD34+ cells is expected, yielding a >99% pure population of EPCs. This system will also have the capability to process multiple, smaller batches at one time using only standard disposable labware for cell- contacting materials. Finally, the functional properties of clinically-relevant EPC populations isolated fluorescently or by ASL will be evaluated through in vitro and in vivo assays. ASL shows considerable promise as a powerful isolation technique that can be applied both clinically for the treatment of acute myocardial infarctions and in individual research settings for the analysis of rare cellular species in blood. This project is significant because in-lab isolation of large volums of cells will enable the widespread use of EPC therapy for improved cardiac function following acute myocardial infarctions. Future directions of this work include the use of ASL in the isolation of other rare cell populations (other progenitor cells, circulating tumor cells) emergingin clinical research, diagnostics, prognostics, and treatment.